Project description:High throughput RNA sequencing For RNA sequencing, F. nucleatum was incubated with 1 mM or 5 mM metformin for 7 hours, when the bacterium were under logarithmic phase. Total RNA of F. nucleatum was stabilized with RNA protect Bacteria Reagent (QIAGEN, Germany) and extracted using a QIAGEN RNeasy kit (QIAGEN, Germany) following the manufacturer’s instructions.
Project description:Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered much attention for its newly discovered prevalence in colorectal and breast cancer tissue. The growing interest in this emerging cancer-associated bacterium sharply contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. Post-transcriptional networks are also unknown, for fusobacteria lack all established small RNA-associated proteins. Here, we present high-resolution global RNA maps for two clinically relevant F. nucleatum subspecies for different growth conditions, and use these to uncover fundamental aspects of fusobacterial gene expression architecture and a previously unknown suite of noncoding RNAs. Developing a new vector for functional analysis of fusobacterial genes, we identify a conserved oxygen-induced small RNA as a post-transcriptional repressor of major porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum to colonize different compartments of the human body.
Project description:Fusobacterium nucleatum is a Gram negative oral bacterial species associated with periodontal disease progression. This species is perhaps best known for its ability to adhere to a vast array of other bacteria and eukaryotic cells. Numerous studies of F. nucleatum have examined various coaggregation partners and inhibitors, but it is largely unknown whether these interactions induce a particular genetic response. We tested coaggregation between F. nucleatum ATCC strain 25586 and various species of Streptococcus in the presence of a semi-defined growth medium containing saliva. We found that this condition could support efficient coaggregation, but surprisingly also stimulated a similar degree of autoaggregation. We further characterized the autoaggregation response, since few reports have examined this in F. nucleatum. After screening several common coaggregation inhibitors, we identified L-lysine as a competitive inhibitor of autoaggregation. We performed a microarray analysis of the planktonic vs. autoaggregated cells and found nearly 100 genes that were affected after only about 60 min. of aggregation. We tested a subset of these genes via real-time RT-PCR and confirmed the validity of the microarray results. Some of these genes were also found to be inducible in cell pellets created by centrifugation. Based upon these data, it appears that autoaggregation activates a genetic program that may be utilized for growth in a high cell density environment, such as the oral biofilm. The study aims to determine the effect of autoaggregation upon the transcriptome. The study contains 2 separate experiments that both measure dispersed (i.e. non-aggregated) vs. aggregated cells and each experiment was performed in duplicate. Samples with no added components other than medium were dispersed, samples containing 25% saliva were aggregated, and samples containing 25% saliva + 50mM L-lysine remained dispersed.
Project description:Fusobacterium nucleatum is a Gram-negative oral bacterial species associated with periodontal disease progression. As periodontal disease progresses, it is known F. nucleatum coaggregated with blood is frequently detected in the gingival crevice. However, it is largely unknown whether these interactions between F. nucleatum and blood induce a particular genetic response. We tested the cultures of F. nucleatum ATCC 25586 with or without blood in a semi-defined growth medium by microarray analysis and found 14 genes that were affected after only about 4hr. of adhered blood. Then, we selected 7 genes that changed significantly and tested these genes via real-time RT-PCR to confirm the validity of the microarray results. As a result, one amino sugar-binding protein on the membrane of F. nucleatum was especially expressed high via both microarray and real-time RT-PCR. Based upon these data, it appears that the protein on the F. nucleatum membrane binds and transfers the amino sugar only under blood conditions. This study aims to determine the effect of blood on the gene expression profiling of F. nucreatum. The study contains 2 separate experiments that both measure the cultures without or with blood. The samples with blood contain 9% and 33% blood.
Project description:The oral commensal Fusobacterium nucleatum can spread to extra-oral sites where it is associated with pathologies as diverse as pre-term birth or cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of RNA regulatory networks that allow this bacterium to adapt to different environmental niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped 5’- and 3’-ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156 nucleotide sRNA previously misannotated as the 5’ UTR of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from most other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5’- and 3’ UTRs in F. nucleatum and expands our understanding of the σE-response in this evolutionarily distant bacterium.
Project description:Fusobacterium nucleatum is a Gram negative oral bacterial species associated with periodontal disease progression. This species is perhaps best known for its ability to adhere to a vast array of other bacteria and eukaryotic cells. Numerous studies of F. nucleatum have examined various coaggregation partners and inhibitors, but it is largely unknown whether these interactions induce a particular genetic response. We tested coaggregation between F. nucleatum ATCC strain 25586 and various species of Streptococcus in the presence of a semi-defined growth medium containing saliva. We found that this condition could support efficient coaggregation, but surprisingly also stimulated a similar degree of autoaggregation. We further characterized the autoaggregation response, since few reports have examined this in F. nucleatum. After screening several common coaggregation inhibitors, we identified L-lysine as a competitive inhibitor of autoaggregation. We performed a microarray analysis of the planktonic vs. autoaggregated cells and found nearly 100 genes that were affected after only about 60 min. of aggregation. We tested a subset of these genes via real-time RT-PCR and confirmed the validity of the microarray results. Some of these genes were also found to be inducible in cell pellets created by centrifugation. Based upon these data, it appears that autoaggregation activates a genetic program that may be utilized for growth in a high cell density environment, such as the oral biofilm.